Structural and Functional Studies of RegB, a New Member of a Family of Sequence-specific Ribonucleases Involved in mRNA Inactivation on the Ribosome

Abstract

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.Control of messenger RNA decay is a key factor in the regulation of gene expression. Indeed, the expression level of a protein is strongly influenced by the concentration of its mRNA, which, in turn, depends on its synthesis and degradation rates (1). Perturbation of mRNA stability can lead to dramatic effects on cell physiology and in some cases can induce cancer (2). However, the way the cell controls the lifetime of its mRNA is still a poorly understood aspect in the biology of both prokaryotic and eukaryotic organisms. In Escherichia coli, it is generally agreed that the initiation of mRNA degradation is induced by an endoribonucleolytic cleavage mediated by ribonucleases E and G with the occasional involvement of RNases III and P (3, 4). These ribonucleases all generate 5Ј-phosphate RNA fragments (RNases E, G, and III are metallo-enzymes; RNase P is a ribozyme), have broad specificity, and are involved in other cellular functions. RNases E and G, for example, participate in the maturation of 9 S and 16 S rRNA (5), and RNase E is necessary for tRNA processing (6).Ribonuclease RegB is radically different from the ribonucleases described above. RegB is involved in the control of the bacteriophage T4 multiplication cycle. It is produced in the early stage of viral infection and facilitates the transition between the early and middle phases of the viral cycle by inactivating early mRNAs (7). RegB cleaves its substrates with high specificity in the middle of the GGAG motif found in the ribosome-binding site of most prokaryotic mRNAs (Shine-Dalgarno sequence). Strikingly, it remains active throughout the entire lytic cycle but does not act on middle or late mRNA (8). In contrast with RNases E, G, III, and P, RegB is only involved in this task and is certainly one of the most specific ribonucleases described to date. It only cleaves GGAG, or to a lesser extent and with reduced efficiency, GGAU sequences (7, 9). Moreover, it does not cleave all such sequences: those present in coding sequences and in middle or late T4 mRNAs are ...

Characterization of Escherichia coli dinJ-yafQ Toxin-Antitoxin System Using Insights from Mutagenesis Data

“…(C) EMSA analysis of DinJ-YafQ(His) 6 protein complex interaction with the palindromic sequences of dinJ-yafQ promoter region. EMSA was performed to detect the interactions of purified DinJ-YafQ(His) 6 RNase (24,33,37,46). Since the structure and the organization of the active site of YafQ is still unknown, we compared several models of YafQ with RelE family RNases with resolved X-ray structures.…”

Spontaneous Dominant Mutations in Chlamydomonas Highlight Ongoing Evolution by Gene Diversification

“…Rather, the degradation of the transcript would depend on a tight contact between the ribosome and NCC2 M , as supported by the limited decrease in petA mRNA observed upon early translation termination, a few nucleotides upstream of the NCC2 M target. This interaction would change the conformation of NCC2 M itself or of its interacting endonuclease, thereby activating nucleolytic activity, as shown for ribosome associated YoeB, RelE, and RegB RNases (Odaert et al, 2007;Neubauer et al, 2009;Feng et al, 2013). By contrast, the degradation of the atpA transcript by NCC1 M does not require ongoing atpA translation.…”

RNA Recognition and Cleavage by Sequence-Specific Endoribonucleases

“…First, RegB, YoeB, RelE, Colicin E5 and Colicin D appear to define a common overall fold despite the modest scores of pairwise structural alignment [34,[38][39][40][41]. This / fold is composed of a central four or five-stranded -sheet and three helices.…”